In early phases of Alzheimer's disease (AD) only specific brain regions are affected. As the disease progresses, neurons in additional regions gradually start expressing signs of pathology and degeneration advances throughout the brain. Understanding the basis for higher vulnerability in specific neurons may point towards the causes of AD and lead to novel therapeutic targets. The two molecular pathological features of AD are excessive accumulation of amyloid-? peptide (A?) in the form of amyloid plaques, and intracellular formation of hyper-phosphorylated tau protein inclusions (neurofibrillary tangles, or NFTs). Neurons from layer II of the entorhinal cortex (ECII) are the first cell type to present with NFTs and neurodegeneration, and are therefore the focus of this research. The absence of a relevant model system remains a serious limitation to AD research: mouse models do not fully recapitulate central molecular events in AD pathology, including neurodegeneration, and currently available human stem cell-derived AD model systems involve nonspecific mixed-populations of neurons[1]?[7], in which cell-specific factors that are potentially crucial for pathological mechanisms of AD might be missing. In this study, an in vitro culture system for human ECII neurons will be developed, using induced pluripotent stem cells (iPSC). ECII neurons have been understudied from a molecular perspective and no culture system, or characterization method exists for these cells. Here, publicly available human genomics data, paired with highly specific bacTRAP molecular profiles of mouse neurons[8] will be employed to gain unprecedented insight into human ECII gene expression. Proteins found to be ECII-specific will be fluorescently tagged in human iPSC, using CRISPR-cas9. Following iPSC differentiation, fluorescent protein expression will be used to monitor and analyze putative ECII cells. TRAP profiling and RNA sequencing will be used to confirm cell identity and to further refine the genetic profile of human ECII cells, in order to characterize master regulators that control cell differentiation. The top master regulator candidates obtained will then be overexpressed in human iPSC to enrich neuronal cultures in ECII neurons. In vitro-differentiated ECII cell identity will be confirmed molecularly, electrophysiologically, histologically, and by examining cell connectivity patterns following targeted transplantation of these cells in the mouse ECII. Finally, this novel culture system will be validated as a relevant model for the study of AD, by implementing the proposed ECII differentiation protocol to iPS cells from healthy individuals and AD patients, or iPSC modified to overexpress proteins with familial AD mutations. The impact of pathogenic A? species on ECII cells in vitro will also be studied and the appearance of pathology, namely NFTs, will be monitored. Furthermore, an ECII-enriched gene, which was recently identified in our lab and was found to be associated with susceptibility to AD in patients will be investigated in this culture system to assess its influence on formation of NFTs, and thus on the vulnerability of human ECII neurons in AD.